Anti-inflammatory compounds

ABSTRACT

This invention relates to the novel pharmaceutical compositions of Formulas (I) and (II) each of which comprises a compound of Formula (I) or (II) and a pharmaceutically acceptable diluent or carrier. 
     This invention also relates to a method of treating or reducing inflammation in a mammal in need thereof, which comprises administering to said mammal an effective amount of a compound or composition of Formula (I) or (II).

FIELD OF THE INVENTION

This invention relates to pharmaceutical compositions and their use asanti-inflammatory agents in mammals.

BACKGROUND OF THE INVENTION

An early event in the response of most inflammatory cells to immunologicactivation and other stimuli is the release of newly formed products(mediators) which alter the function and biochemistry of surroundingcells and tissues. The ensuing biological responses, as well as much ofthe pathogenesis which is attributed to inflammation and allergy, arethought to be dependent on the effects that these newly-formed mediatorshave on adjacent cells within the inflammatory region.

In the last 20 years, it has become apparent that lipid mediators areamong the most potent and important products which are generated duringinflammatory reactions. The synthesis of most lipid mediators isinitiated by the specific cleavage of complex phospholipid moleculeswhich contain arachidonate at their sn-2 position. Arachidonic acid ispredominantly found in the sn-2 position of phospholipids afterredistribution by transacylases and its release by sn-2 acylhydrolasesfrom phospholipids represents the rate-limiting step in the formation ofeicosanoids (leukotrienes, prostaglandins and thromboxanes) and otherhydroxylated fatty acids. As arachidonic acid is released, it is thenconverted to oxygenated derivatives by at least two enzymatic systems(lipoxygenase and/or cyclooxygenase). Concomitant with arachidonaterelease, lysophospholipids are formed. One of these lyso phospholipids,1-alkyl-2-lyso-sn-glycero-3-phosphocholine, is then acetylated to formplatelet-activating factor (PAF). Each of the cell types involved in theinflammatory response produce and secrete a unique subset of lipidmediators. The quantities and nature of the metabolites depend on whichenzymes and precursor phospholipid pools are available to inflammatorycells.

Once lipid mediators such as PAF and eicosanoids are formed by theaforementioned pathways, they induce signs and symptoms observed in thepathogenesis of various inflammatory disorders. Indeed, thepathophysiological activity of arachidonic acid (and its metabolites) iswell known to those skilled in the art. For example, these mediatorshave been implicated as having an important role in allergy, asthma,anaphylaxis, adult respiratory distress syndrome, reperfusion injury,inflammatory bowel disease, rheumatoid arthritis, endotoxic shock, andcardiovascular disease. Aalmon et al., Br. Med. Bull (1978) 43:285-296;Piper et al., Ann. New York Acad. Sci. (1991) 629:112-119; Holtzman, Am.Rev. Respir. Dis. (1991) 143:188-203; Snyder, Am. J. Physiol. CellPhysiol. (1990) 259:C697-C708; Prescott et al., J. Biol. Chem. (1990)265:17381-17384.

Similar to arachidonate products, PAF is a potent proinflammatorymediator produced by a variety of cells. In vitro, PAF stimulates themovement and aggregation of neutrophils and the release therefrom oftissue-damaging enzymes and oxygen radicals. PAF has also beenimplicated in activation of leukocytes, monocytes, and macrophages.These activities contribute to the actions of PAF as having(pathological) physiological activity in inflammatory and allergicresponses. PAF has also been implicated in smooth muscle contraction,pain, edema, hypotensive action, increases in vascular permeability,cardiovascular disorders, asthma, lung edema, endotoxin shock, and adultrespiratory distress syndrome. PAF elicits these responses eitherdirectly through its own cellular receptor(s) or indirectly by inducingthe synthesis of other mediators.

Accordingly, a method which antagonises the production of freearachidonic acid, its metabolites or PAF will have clinical utility inthe treatment of a variety of allergic, inflammatory and hypersecretoryconditions such as asthma, arthritis, rhinitis, bronchitis andurticaria, as well as reperfusion injury and other disease involvinglipid mediators of inflammation. Many published patent applications orissued US patents exist which describe various compounds having utilityas PAF or eicosanoid antagonists. Such patents include U.S. Pat. Nos.4,788,205, 4,801,598, 4,981,860, 4,992,455, 4,983,592, 5,011,847,5,019,581 and 5,002,941.

Phospholipase A2's (PLA₂, (EC 3.1.1.4)) are responsible for theliberation of arachidonic acid from the sn-2 position of phospholipid.They are therefore thought to play an important role in the pathogenesisof inflammation and possibly in immunological dysfunction, both as acell associated enzyme as well as an extracellular soluble enzyme. Lowmolecular weight, mammalian Type II 14 kDa PLA₂ has been wellcharacterized and is known to exist in both an extracellular form ininflammatory fluids (Kramer et at., J. Biol. Chem., 264:5768-5775(1989)) and in a cell associated form (Kanda et al., Biochemical andBiophysical Research Communications, 163:42-48 (1989)) and has beenfound in a variety of cells and tissues or extracellularly when releasedin response to antigenic activators or pro-inflammatory mediators suchas Interleukin (IL)-1, IL-6 or tumor necrosis factor (TNF). Its presencein such inflammatory fluids, tissue exudates or serum has thereforeimplicated Type II14 kDa-PLA₂ 's role in inflammation (Vadas, et at.,(1985) Life Sci. 36, 579-587; and Seilhamer, et at., (1989) J. Biol.Chem. 264, 5335-5338). Recently, the elevated serum levels of PLA₂activity during an inflammatory insult has been attributed to cytokineinduction of acute phase protein release from liver, of which the 14kDa-PLA₂ is suggested to be a part (Crowl, et al., (1991) J. Biol. Chem.266, 2647-2651). In addition, soluble PLA₂ activity is markedly elevatedin the serum and synovial fluid of patients with rheumatoid arthritis(Stefanski et at., J. Biochem. 100:1297-303 (1986). Furthermore,increasing serum PLA₂ levels have been shown to positively correlatewith clinical severity (Bomalaski and Clark, Arthritis and Rheumat.36:190-198 (1993)). Various inhibitors of PLA₂ have been described inpublications and in U.S. Patents. See for instance U.S. Pat. Nos.4,959,357; 4,933,365; 5,208,223; 5,208244; Marshall et al., J.Rheumatology 18:1 (1991 ); Marshall et at., Phospholipase A2, Ed. PyuWong, Plenum Press, New York (1990) pages 169-181; Wilkerson, et at.,Eur. J. Med. Chem., 26:667, 1991 and Wilkerson, AntiinflammatoryPhospholipase A₂ Inhibitors, Drugs of the Future, Vol. 15, No. 2 p139-148(1990). Accordingly, as PLA₂ is important in the liberation ofarachidoninc acid from phospholipid and may also play a role in thegeneration of PAF via lysophospholipid formation, inhibition of such anenzyme would be useful for the treatment of disease states causedthereby.

There are many novel forms of phospholipase A₂ 's which have recentlybeen discovered. For the purposes herein, members of the sn-2acylhydrolase family of PLA₂ 's are divided into low and high molecularweight enzymes be it from mammalian, or non-mammalian sources. Lowmolecular weight PLA₂ 's will generally have a molecular weight in therange of 12,000 to 15,000. High molecular weight will be in the range of30,000 or 56,000 kDa to 110,000 by SDS electrophoresis analysis.

A high molecular weight, cytosolic 85 kDa PLA₂ has been isolated andcloned from the human moncytic cell line, U937 (Clark et al., Proc.Natl. Acad. Sci., 87:7708-7712, 1990). The cell-associated Type II-14kDa-PLA₂ in cell lipid metabolism was thought to be the key ratelimiting enzyme in lipid mediator formation, until the recentidentification of this cell-associated but structurally distinct 85 kDasn-2 acylhydrolase, (Clark, et al., supra); and Kramer, et al., (1991)J. Biol. Chem. 266, 5268-5272. Like the Type II-14 kDa enyzme, thisenzyme is active at neutral pH and Ca²⁺ -dependent, but in contrastexhibits a preference for AA in the sn-2 position of phospholipidsubstrate and migrates from the cytosol to the membrane in a Ca²⁺-dependent manner and is regulated by phosphorylation (Kramer et al., J.Biol. Chem., 266:5268-5272 (1991). The 85 kDa-PLA₂ is also distinct from14 kDa-PLA₂ s and Ca²⁺ -independent PLA₂ as demonstrated by differentbiochemical characteristics such as stability of the 85 kDa-PLA₂ to DTT,instability to heat and the lack of inhibition by a phosphonatephospholipid TSA inhibitor of 14 kDa-PLA₂. In addition, 85 kDa-PLA₂ hasbeen shown to possess a lysophospholipase A₁ activity which is notobserved with the 14 kDa-PLA₂ s. The 85 kDa enzyme is similar to themyocardial Ca2+-independent PLA₂ (Hazen and Gross, Circ. Res. 70:486-495(1992)) in that Ca²⁺ is not required for catalysis and DTNB inhibitionis observed. However, 85 kDa-PLA₂ is not inhibited by the suicideinactivator bromoenol lactone, suggesting that the enzyme is distinctfrom the myocardial enzyme as well. These characteristics make the 85kDa-PLA₂ a candidate for participation in the liberation of AA fromphospholipid stores for subsequent metabolism to lipid mediators. Boththe cytosolic 85 kDa PLA₂ and a cell associated Type II 14 kDa PLA₂ havebeen found in the human immune cells such as monocyte, neutrophil andplatelet (Marshall and Roshak, Biochem. Cell Biol. 71:331-339 (1993)).As noted above most of the cellular lipid mediators found elevated in avariety of inflammatory fluid were formed in response to non-pancreaticPLA₂ action.

Since arachidonate-containing phospholipids are the key precursors for abroad range of lipid mediators it would not be surprizing that,inflammatory cells would treat these phospholipids differently thanother fatty acid-containing phospholipids. In particular, there areenzymes which control the mount of arachidonate in differentphospholipid pools and these enzymes are tightly regulated to maintainarachidonate homeostasis. The movement of arachidonate into and from allphospholipids was originally thought to be exclusively by CoenzymeA-dependent acyl transferase activitites. Holub et al., Adv. Lipid Res.,16:1-125 (1978); Lands et al., In The Enzymes of Biological Membranes,ed. Martonosi, A., pp. 3-85, Plenum Press, New York, 1976. However, ithas now been demonstrated that an enzyme, Coenzyme A-independenttranscylase (CoA-IT), is involved in the movment of 20 carbon higherunsaturated fatty acids, particularly arachidonate, into particular(1-alkyl- and 1-alkenyl)phospholipid pools. These are the phospholipidpools of arachidonate that are preferentially mobilized during cellactivation and utilized for eicosanoid and PAF biosynsthesis,respectively.

CoA-IT has a specificity for certain phospholipids as donor and acceptormolecules. The fatty acid transferred is long chained and unsaturated,and almost exclusively arachidonate. Other fatty acids such as the 16:0,18:1 or 18:2 are not moved into the sn-2 position of alkyl and 1-alkenylphospholipid pools by CoA-IT. The specificity of CoA-IT is in directcontrast to many other CoA-dependent acylation activities which acylatea wide variety of lysophospholipids with no selectivity forarachidonate.

Accordingly, as CoA-IT is involved in arachidonic acid and phospholipidmetabolism, inhibition of such an enzyme would be useful for thetreatment of inflammatory, allergic and hypersecretory conditions ordisease states caused thereby. Therefore, a method by which CoA-IT isinhibited will consequently and preferentially decrease the arachidonatecontent of 1-alkyl- and 1-alkenyl-linked phospholipids and willtherefore decrease the production of pro-inflammatory mediators such asfree arachidonic acid, prostaglandins, leukotriene and PAF during aninflammatory response.

SUMMARY OF THE INVENTION

This invention relates to a pharmaceutical compositions of Formula (I)comprising a compound of Formula (I), or pharmaceutically acceptablesalt thereof and a pharmaceutically acceptable diluent or carrier.

The present invention also provides for a pharmaceutical compositioncomprising a pharmacuetically acceptable carrier or diluent and acompound, or pharmaceutically acceptable salt thereof, of Formula (II).

This invention also relates to a method of treating or reducinginflammation in a mammal in need thereof, which comprises administeringto said mammal an effective amount of a compound or composition ofFormula (I) or Formula (II).

This invention also relates to a method of treating disease or disordersmediated by PLA₂ and/or CoA-IT, free arachidonic acid, its metabolitesand/or PAF by administering to a patient in need thereof, an effectiveamount of a compound of Formula (I) or Formula (II).

This invention also relates to a method of treating disease or disordersmediated by phospholipase A₂ and/or CoA-IT, by administering to apatient in need thereof, an effective amount of a compound orcomposition of Formula (I) or Formula (II).

One aspect of the present invention is a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier or diluent and acompound represented by the structure corresponding to the formula:##STR1## wherein R₄ is a phenyl subsiuted one to two times independentlywith chlorine or CF₃ ;

R₁ is chlorine;

R₂ is hydrogen or chlorine;

R₃ is chlorine or CF₃ ;

provided that when R₁ and R₂ are both chlorine then R₃ is CF₃ ;

and pharmaceutically acceptable salts thereof.

Another aspect of the present invention is a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier or diluent and acompound represented by the structure corresponding to the formula:##STR2## wherein R₄ is a phenyl substituted one to two timesindependently with chlorine or CF₃ or R₄ is a disubstituted phenylsubstituted once by chlorine or CF₃ and once by 3-chlorophenoxy or a4-chlorophenoxy group;

R₁ is chlorine or --C((CH₃)₂)CH₂ CH₃ ;

R₂ is hydrogen, chlorine or methyl;

R₂ ' is hydrogen or chlorine;

R₃ is chlorine or CF₃ ;

provided that

a) when R₂ is methyl, then R₁ and R₃ are both chlorine and R₄ is a 3-CF₃-4-chlorophenyl, 3,4-dichlorophenyl, or 2-methyl-6-chlorophenyl;

b) when R₁ is t-amyl then R₂ and R₂ ' are hydrogen;

c) when R₁ is t-amyl, R₂ and R₂ ' are hydrogen, and R₃ is CF₃, then R₄is 3-CF₃ -phenyl, 3,5-bis(trifluoromethyl)phenyl or 4-chloro-3-CF₃phenyl;

d) when R₂ ' is chlorine, then R₂ is hydrogen, R₁ and R₃ are bothchlorine and R₄ is 2-methyl-4-chlorophenyl;

e) when R₁ and R₃ are chlorine, and R₂ and R₂ ' are hydrogen, then R₄ is2,5-dichlorophenyl, 3,5-di-trifluoromethyl phenyl, 2-chloro-5-CF₃-phenyl, 3-CF₃ -4-chlorophenyl, 3,4-dichlorophenyl, 2,3-dichlorophenyl,4-chlorophenyl, 3-trifluoromethylphenyl, 3-chloro-4-(4-chlorophenoxy),3-CF₃ -6-(4-chlorophenoxy), 3-chloro-6-(4-chlorophenoxy);

f) when R₁, R₂ and R₃ is chlorine, then R₄ is 2-chloro-5-CF₃ -phenyl or3-CF₃ -4-chlorophenyl;

g) when R₁ and R₂ are chlorine, and R₃ is CF₃, then R₄ is 4-chloro-3-CF₃phenyl;

and pharmaceutically acceptable salts thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a novel method of treatinginflammatory disease in a mammal in need thereof by administering tosaid mammal an effective amount of a compound according to Formula (I)or Formula (II). The compounds of Formula (I) and (II) may selectivelyinhibit the PLA₂ enzyme, the CoA-IT enzyme or both. Inhibition of eitheror both enzymes will result in the treatment of inflammatory occurrencesin mammals. Inflammatory states in mammals may include, but are notlimited to, allergic and asthmatic manifestations, dermatologicaldiseases, inflammatory diseases, collagen diseases, reperfusion injuryand stroke. Treatment of both acute and chronic diseases are possible.Preferred diseases for treatment are arthritis, asthma, allergicrhinitis, inflammatory bowel disease (IBD), psoriasis, reperfusioninjury and stroke. For the purposes herein, the compounds of Formula (I)and (II) are preferential and selective inhibitors of the low molecularweight PLA₂ enzyme.

For purpoeses herein, the compounds of the generic formulas (I) and(II), for the (R₂)_(m) term provisos, etc. are numbered by the point ofattachment to the ether or thioether. This point of attachment is theone position, the R₁ term is the 2-position, etc. The nomenclature usedfor naming the pecifically exemplified compounds, such as common orIUPAC corresponds to actual rules of nomenclature as is uneffected bythe generic formula herein.

Specifically exemplified compounds of Formula (I) are:

4,5-Dichloro-2-[4-trifluoromethyl-2-(3-trifluoromethyl-4-chlorophenyl)ureido)phenyl)thio]benzenesulfonicacid;

4,5-Dichloro-2-[3-(3,4-dichlorophenyl)ureido]-4-trifluoromethylphenyl)thiobenzenesulfonicacid;

5-Chloro-2-[2-(3-(3,5-di-trifluoromethylphenyl)ureido)-4-trifluoromethylphenyl)thio]benzenesulfonicacid;

5-Chloro-2-[(4-chloro-2-(3-(5-chloro-3-trifluoromethylphenyl)ureido)phenyl)thio]benzenesulfonicacid;

5-Chloro-2-[(4-chloro-2-[3-(2,4-dichlorophenyl)ureido)phenyl)thio]benzenesulfonicacid;

5-Chloro-2-[(4-chloro-2-(3-(4-chloro-3-trifluoromethylphenyl)ureido)phenyl)thio]benzenesulfonicacid.

Specifically exemplified compounds of Formula (II) are:

5-Chloro-2-[(4-chloro-2-(3(2,5-dichlorophenyl)ureido)phenyloxy]benzenesulfonicacid;

4,5-Dichloro-2-[4-chloro-2-[[[[2-chloro-5-(trifluoromethyl)phenyl]-amino]carbonyl]amino]phenoxy]benzenesulfonicacid;

5-Chloro-2-[4-chloro-2-[[[[4-chloro-3-(trifluoromethyl)phenyl]amino]carbonyl]amino]phenoxy]-4-methylbenzenesulfonic acid;

5-Chloro-2[(4chloro-2-[3(3,4-dichlorophenyl)ureido)phenyloxy]-4-toluenesulfonicacid;

2-[2-[3-(4-Chloro-2-tolyl)ureido]-4-chlorophenoxy]-3,5-dichlorobenzenesulfonicacid;

2-[2-[3-(4-Chloro-3-trifluoromethylphenyl)ureido]-4-trifluoromethylphenoxy]-4,5-dichlorobenzenesulfonic acid;

2-[2-[[[[3,5-Bis(trifluoromethyl)phenyl]amino]carbonyl]amino]-4-chlorophenoxy]-5-chlorobenzeneacid;

5-(1,1-Dimethylpropyl)-2-[4-(trifluoromethyl)-2-[[[[3-(trifluoromethyl)phenyl]amino]carbonyl]amino]-phenoxy]benzenesulfonicacid;

2-[2-[3-(4-Chloro-3-trifluoromethylphenyl)ureido]-4-trifluoromethylphenoxy]-5-(1,1-dimethylpropyl)benzenesulfonicacid;

2-[2-[[[[3,5-Bis(trifluoromethyl)phenyl]amino]carbonyl]amino]-4-(trifluoromethyl)phenoxy]-5-(1,1-dimethylpropyl)benzenesulfonicacid;

4,5-Dichloro-2-[4-chloro-2-[[[[4-chloro-3-(trifluoromethyl)phenyl]amino]carbonyl]amino]phenoxy]benzenesulfonicacid;

5-Chloro-2-[4-chloro-2-[[[[2-chloro-5-(trifluoromethyl)phenyl]amino]carbonyl]amino]phenoxy]benzenesulfonicacids.

5-Chloro-2-[4-chloro-2-[[[[4-chloro-3-(trifluoromethyl)phenyl]amino]carbonyl]amino]phenoxy]benzenesulfonicacid;

5-Chloro-2-(4-chloro-2-(3,4-dichlorophenylaminocarbonylaminophenoxy)benzenesulfonic acid;

5-Chloro-2-(4-chloro-2-(2,3-dichlorophenylaminocarbonylaminophenoxy)benzenesulfonic acid;

5-Chloro-2-(4-chloro-2-(4-chlorophenylaminocarbonylaminophenoxy)benzenesulfonic acid;

5-Chloro-2-(4-chloro-2-(3-trifluoromethylphenylaminocarbonylaminophenoxy)benzenesulfonic acid;

5-Chloro-2-[4-chloro-2-(2-chloro-5-trifluoromethylphenylamino)carbonylamino]phenoxybenzenesulfonic acid;

5-Chloro-2-(4-chloro-2-(3-chloro-4-(4-chlorophenoxy)-phenylaminocarbonylamino)phenyoxybenzenesulfonic acid;

5-Chloro-2-(4-chloro-2-(2-(3-chlorophenoxy)-5-trifluoromethylphenylaminocarbonylamino)phenyoxybenzenesulfonic acid;

5-Chloro-2-[4-chloro-2-(5-chloro-2-(4-chlorophenoxy)phenylaminocarbonylamino)phenoxy]benzenesulfonic acid.

Another aspect of the present invention for use as PLA₂ and/or CoA-ITinhibitors in the treatment of inflammatory conditions am the novelcompounds and pharmacuetically acceptable salts thereof:

5-(1,1-Dimethylpropyl)-2-[4-(trifluoromethyl)-2-[[[[3-(trifluoromethyl)phenyl]amino]carbonyl]amino]-phenoxy]benzenesulfonicacid;

2-[2-[3-(4-Chloro-3-trifluoromethylphenyl)ureido]-4-trifluoromethylphenoxy]-5-(1,1-dimethylpropyl)benzenesulfonicacid; and

2-[2-[[[[3,5-Bis(trifluoromethyl)phenyl]amino]carbonyl]amino]-4-(trifluoromethyl)phenoxy]-5-(1,1-dimethylpropyl)benzenesulfonicacid

Preferably the pharmaceutically acceptable salts are of the alkalimetals, such as sodium.

SYNTHETIC CHEMISTRY

Compounds of Formula (I) and (II) can be readily prepared by one skilledin the art in an analagous manner to the synthesis as indicated below.Alternatively, the compounds specifically exemplified above, except for2-[2-[3-(4-Chloro-3-trifluoromethylphenyl)ureido]-4-trifluoromethylphenoxy]-5-(1,1-dimethylpropyl)benzenesulfonicacid;5-(1,1-Dimethylpropyl)-2-[4-(trifluoromethyl)-2-[[[[3-(trifluoromethyl)phenyl]amino]carbonyl]amino]-phenoxy]benzenesulfonicacid and2-[2-[[[[3,5-Bis(trifluoromethyl)phenyl]amino]carbonyl]amino]-4-(trifluoromethyl)-phenoxy]-5-(1,1-dimethylpropyl)benzenesulfonicacid can be purchased through the BADER catalog of ALDRICH ChemicalCompany upon request.

Without further elaboration, it is believed that one skilled in the artcan, using procedures analagous to those described herein, utilize thepresent invention to its fullest extent. The invention will now bedescribed by reference to the following examples which are merelyillustrative and are not to be construed as a limitation of the scope ofthe present invention. Temperatures are recorded in degrees centigradeunless otherwise noted.

Example 12-[2-[3-(4-Chloro-3-(trifluoromethyl)phenyl]ureido]-4-(trifluoromethyl)phenoxy]-4,5-dichlorobenzenesulfonicacid, sodium salt

a) 2-(2-Nitro-4-trifluoromethylphenoxy)-4,5-dichlorobenzene

A mixture of 3,4-dichlorophenol (25 gram (hereinafter g), 0.153 mol),4-chloro-3-nitrobenzo-trifluoride (52 g, 0.23 mol) and potassiumcarbonate (63 g, 0.459 mol) in dimethylformamide (450 mL)was stirredunder argon at 120° C. for 24 h. The reaction mixture was filtered andthe filtrate evaporated. The residue was dissolved in ethyl acetate,washed with water, brine, dried over anhydrous magnesium sulfate,filtered and evaporated. The residue was flash chromatographed (silicagel, methylene chloride/hexane), to give the title compound as a yellowoil.

b) 2-(2-Nitro-4-trifluoromethylphenoxy)-4,5-dichlorobenzenesulfonicacid, ammonium salt

Fuming sulfuric acid (8 mililiter (hereinafter mL) was cooled to -20° C.and 2-(2-nitro-4-trifluoromethyl-phenoxy)-4,5-dichlorobenzene (8 g, 22.8mmol) in methylene chloride (8 mL) was added to the solution. Themixture was stirred for 15 min and then quenched in ice. Extraction withethyl acetate followed by flash chromatography (silica gel, methylenechloride/isopropanol/ammonium hydroxide) gave the title compound. ¹ HNMR (400 MH_(z), CD₃ OD) δ8.31 (d, 1H), 8.11 (s, 1H), 7.87 (dd, 1H), 7.4(s, 1H), 7.16 (d, 1H).

c) 2-(2-Amino-4-trifluoromethylphenoxy)-4,5-dichlorobenzenesulfonic acid

The 2-(2-nitro-4-trifluoromethylphenoxy)-4,5-dichlorobenzenesulfonicacid, ammonium salt (5 g, 11.2 mmol) was dissolved in acetic acid (75mL). The mixture was diluted with water (75 mL), and a 20 % aqueoussolution of titanium trichloride (65 mL) was added. After aqueous workupthe crude product was flash chromatographed (silica gel, methylenechloride/ethanol/ammonium hydroxide) to give the title compound. ¹ H NMR(400 MH_(z), CD₃ OD) δ8.02 (s, 1H), 7.17 (d, 1H), 7.12 (d, 1H), 6.94(dd, 1H), 6.87 (s, 1H).

d)2-[2-[3(4-Chloro-3-(trifluoromethyl)phenyl]ureido]-4-(trifluoromethyl)phenoxy]-4,5-dichlorobenzenesulfonicacid, ammonium salt

A solution of2-(2-amino-4-trifluoromethylphenoxy)-4,5-dichlorobenzenesulfonic acid (2g, 4.8 mmol) and 4-chloro-3-trifluoromethylphenyl isocyanate (1.11 g, 5mmol) in pyridine (20 mL) was stirred under argon for 72 hours(hereinafter h). The solvent was evaporated and the residue was flashchromatographed (silica gel, methylene chloride/ethanol/ammoniumhydroxide) to give the title compound. ¹ H NMR (400 MH_(z), CD₃ OD)δ8.61 (d, 1H), 8.08 (s, 1H), 7.95 (d, 1H), 7.60 (dd, 1H), 7.43 (d, 1H),7.30-7.36 (m, 2H), 7.10 (s, 1H).

e)2-[2-[3-(4-Chloro-3-(trifluoromethyl)phenyl]ureido]-4-(trifluoromethyl)phenoxy]-4,5-dichlorobenzenesulfonicacid, sodium salt

A mixture of2-[2-[3-(4-chloro-3-(trifluoromethyl)phenyl]ureido]-4-(trifluoromethyl)phenoxy]-4,5-dichlorobenzenesulfonicacid, ammonium salt (2.1 g, 3.28 mmol) and sodium bicarbonate (0.331 g,3.94 mmol) in methanol (20 mL) and water (2 mL) were stirred under argonfor 30 min. The solvents were evaporated, and the residue was flashchromatographed (C₁₈ reverse phase, MeOH/H₂ O) to give the titlecompound after lyophilization. MS (ES) m/e 643.8 [M+H]⁺.

EXAMPLE 22-[2-[3-[3,5-Bis(trifluoromethyl)phenyl]ureido]-4-trifluoromethylphenoxy]-5-(1,1-dimethylpropyl)benzenesulfonicacid, sodium salt

a) 2-(2-nitro-4-trifluoromethylphenoxy)-4-(1,1-dimethylpropyl)benzene

A mixture of 4-(1,1-dimethylpropyl)phenol (821 mg, 0.005 mol),4-bromo-3-nitrobenzotrifluoride (1.35g, 0.005 mol) potassium carbonate(1.38g, 0.010 mol) and cuprous oxide (143 mg, 0.001 mol) indimethylformamide (25 ml) was stirred under argon at 150° C. for sixteenhours. The solvent was evaporated and the residue partitioned betweenethyl acetate and water, the layers separated, dried over MgSO₄ andevaporated. The crude product flash chromatographed (silica gel, ethylacetate/hexane) to give the title compound as a yellow oil. ¹ H NMR (250MH_(z), CDCl₃) ∂8.20 (d, 1H), 7.70 (dd, 1H), 7.45 (m, 2H), 7.05 (m, 3H),1.65 (q, 3H), 1.30 (s, 6H), 0.65 (t, 2H).

b)2-(2-Nitro-4-trifluoromethylphenoxy)-5-(1,1-dimethylpropyl)benzenesulfonicacid ammonium salt

Fuming sulfuric acid (1 ml) was added to2-(2-nitro-4-trifluoromethylphenoxy)-4-(1,1-dimethylpropyl)benzene (770mg, 0.0022 mol) and stirred for forty-five minutes, quenched in ice,extracted with ethyl acetate, dried over MgSO₄ and evaporated. Theresidue was flash chromatographed (silica gel, methylenechloride/isopropanol/ammonium hydroxide) to give the title compound. ¹ HNMR (250 MH_(z), CD₃ OD) ∂7.95 (dd, 2H), 7.65 (dd, 1H), 7.45 (dd, 1H),7.00 (d, 1H), 6.80 (d, 1H), 1.65 (q, 3H), 1.26 (s, 6H), 0.65 (t, 2H).

c)2-(2-Amino-4-trifluoromethylphenoxy)-5-(1,1-dimethylpropyl)benzenesulfonicacid

2-(2-Nitro-4-trifluoromethylphenoxy)-5-(1,1-dimethylpropyl)benzenesulfonicacid, ammonium salt (620 mg) was mixed in ethyl acetate (50 ml) andpalladium on carbon (202 mg) was added under argon. The mixture washydrogenated in a Parr bottle at 55 psi for two hours. The sample wasfiltered to remove the catalyst, mixed with methanol and degassed forseveral hours to yield the title compound ¹ H NMR (250 MH_(z), CD₃ OD)∂7.95 (d, 1H), 7.30 (dd, 1H), 7.05 (dd, 2H), 6.85 (dd, 1H), 6.75 (d,1H), 1.65 (q, 3H), 1.25 (s, 6H), 0.65 (t, 2H).

d)2-[2-[3-[3,5-Bis(trifluoromethyl)phenyl]ureido]-4-trifluoromethylphenoxy]-5-(1,1-dimethylpropyl)benzenesulfonicacid, ammonium salt

2-(2-Amino-4-trifluoromethylphenoxy)-5-(1,1-dimethylpropyl)benzenesulfonicacid (260 mg, 0.0006 mol) and 3,5-bis(trifluoromethyl)phenyl isocyanate(130.9 ml, 0.0005 mol) were mixed in pyridine (5 ml), under argon, forsixteen hours at room temperature. The solvent was evaporated and theresidue flash chromatographed (silica gel, methylenechloride/isopropanol/ammonium hydroxide), dried and evaporated to givethe title compound. ¹ H NMR (250 MHz, DMSO) ∂9.20 (s, 1H), 8.90 (s, 1H),7.90 (s, 1H), 7.50 (s, 2H), 7.25 (d, 1H), 7.05 (s, 1H), 6.80-6.30 (m,4H), 1.0 (q, 3H), 0.065 (s, 6H), 0.045 (d, 2H).

e)2-[2-[3-[3,5-Bis(trifluoromethyl)phenyl]ureido]-4-trifluoromethylphenoxy]-5-(1,1-dimethylpropyl)benzenesulfonicacid, sodium salt

A mixture of2-[2-[3-[3,5-bis(trifluoromethyl)phenyl]ureido]-4-trifluoromethylphenoxy]-5-(1,1-dimethylpropyl)benzenesulfonicacid, ammonium salt (254 mg, 0.0004 mol) and sodium bicarbonate (96 mg,0.0011 mol) in methanol and water was stirred under argon for an hour.The solvents were evaporated and the residue was flash chromatographed(C₁₈ reverse phase, MeOH/H₂ O) to give the title compound. MS (FAB) m/e681 [M+H]⁺.

EXAMPLE 32-[2-[3-(4-Chloro-3-trifluoromethylphenyl)ureido]-4-trifluoromethylphenoxy]-5-(1,1-dimethylpropyl)benezenesulfonicacid, sodium salt

a) 2-(2-Nitro-4-trifluoromethylphenoxy)-4-(1,1-dimethylpropyl)benzene

Following the procedure of Example 2(a) the title compound was made. ¹ HNMR (250 MH_(z), CDCl₃) ∂8.20 (d, 1H), 7.70 (dd, 1H), 7.45 (m, 2H), 7.05(m, 3H), 1.65 (q, 3H), 1.30 (s, 6H), 0.65 (t, 2H).

b)2-(2-Nitro-4-trifluoromethylphenoxy)-5-(1,1-dimethylpropyl)benzenesulfonicacid, ammonium salt

Following the procedure of Example 2(b) the title compound was made. ¹ HNMR (250 MH_(z), CD₃ OD) ∂7.95 (dd, 2H), 7.65 (dd, 1H), 7.45 (dd, 1H),7.00 (d, 1H), 6.80 (d, 1H), 1.65 (q, 3H), 1.26 (s, 6H)), 0.65 (t, 2H).

c)2-(2-Amino-4-trifluoromethylphenoxy)-5-(1,1-dimethylpropyl)benzenesulfonicacid

Following the procedure of Example 2(c) the title compound was made. ¹ HNMR (250 MH^(z), CD₃ OD) ∂7.95 (d, 1H), 7.30 (dd, 1H), 7.05 (dd, 2H),6.85 (d, 1H), 6.75 (d, 1H), 1.65 (q, 3H), 1.25 (s, 6H), 0.65 (t, 2H).

d)2-[2-[3-(4-Chloro-3-trifluoromethylphenyl)ureido]-4-trifluoromethylphenoxy-5-(1,1-dimethylpropyl)benzenesulfonicacid, ammonium salt

Following the procedure of Example 2(d) except substituting4-chloro-3-trifluoromethylphenylisocyanate for3,5-bis(trifluoromethyl)phenylisocyanate the title compound was made. ¹H NMR (250 MHz, CD3OD) ∂8.55 (d, 1H), 7.95 (dd, 2H), 7.60 (dt, 1H),7.49-7.20 (m, 4H), 6.89 (d, 1H), 1.60 (q, 3H), 1.20 (s, 6H), 0.65 (t,2H).

e)2-[2-[3-(4-Chloro-3-trifluoromethylphenyl)ureido-4-trifluoromethylphenoxy-5-(1,1-dimethylpropyl)benzenesulfonicacid, sodium salt

Following the procedure of Example 2 (e) except substituting2-[2-[3-(4-chloro-3-trifluoromethylphenyl)ureido]-4-trifluoromethylphenoxy-5-(1,1-dimethylpropyl)benzenesulfonicacid, ammonium salt for2-[2-[3-[3,5-bis(trifluoromethyl)phenyl]ureido]-4-trifluoromethylphenoxy]-5-(1,1-dimethylpropyl)benzenesulfonicacid, ammonium salt. MS (FAB) m/e 669 [M+Na]⁺.

EXAMPLE 44,5-Dichloro-2-[3-(3,4-dichlorophenyl)ureido]-4-trifluoromethylphenyl)-thiobenzenesulfonicacid

The title compound was purchased commercially. The title compound canalso be made in an analogous manner to that of Example 1 above exceptusing 3,4-dichlorothiophenol in place of 3,4-dichlorophenol and3,4-dichlorophenylisocyanate in place of4-chloro-3-trifluoro-methylphenylisocyanate.

EXAMPLE 54,5-Dichloro-2-[4-trifluoromethyl-2-(3-trifluoromethyl-4-chlorophenyl)ureido)phenyl)thio]benzenesulfonicacid

The title compound was purchased commercially. The title compound canalso be made in an analogous manner to that of Example 1 above exceptusing 3,4-dichlorothiophenol in place of 3,4-dichlorophenol.

EXAMPLE 65-Chloro-2-[2-(3-(3,5-di-trifluoromethylphenyl)ureido)-4-trifluoromethylphenyl)thio]benzenesulfonicacid

The title compound was purchased commercially. The title compound canalso be made in an analogous manner to that of Example 1 above exceptusing 4-chlorothiophenol in place of 3,4-dichlorophenol, and3,5-bis(trifluoromethyl)phenyl isocyanate in place of4-chloro-3-trifluoromethylphenyl isocyanate.

EXAMPLE 75-Chloro-2-[(4-chloro-2-((3-(5-chloro-3-trifluoromethylphenyl)ureido)phenyl)thio]benzenesulfonicacid

The title compound was purchased commercially. The title compound canalso be made in an analogous manner to that of Example 1 above exceptusing 4-chlorothiophenol in place of 3,4-dichlorophenol, 2,5dichloronitrobenzene in place of 4-chloro-3-nitrobenzotrifluoride, and2-chloro-5-trifluoromethylphenylisocyanate in place of4-chloro-3-trifluoromethylphenylisocyanate.

EXAMPLE 85-Chloro-2-[(4-chloro-2-[3-(2,4-dichlorophenyl)ureido)phenyl)thio]benzenesulfonicacid

The title compound was purchased commercially. The title compound canalso be made in an analogous manner to that of Example 1 above exceptusing 4-chlorothiophenol in place of 3,4-dichlorophenol, 2,5dichloronitrobenzene in place of 4-chloro-3-nitrobenzotrifluoride, and2,4-dichlorophenylisocyanate in place of4-chloro-3-trifluoromethylphenyl isocyanate.

EXAMPLE 95-Chloro-2-[(4-chloro-2-(3-(4-chloro-3-trifluoromethylphenyl)ureido)phenyl)thio]benzenesulfonicacid

The title compound was purchased commercially. The title compound canalso be made in an analogous manner to that of Example 1 above exceptusing 4-chlorothiophenol in place of 3,4-dichlorophenol and 2,5dichloronitrobenzene in place of 4-chloro-3-nitrobenzotrifluoride.

EXAMPLE 105-Chloro-2-[(4-chloro-2-(3-(2,5-dichlorophenyl)ureido)phenyloxy]benzenesulfonicacid

The title compound was purchased commercially. The title compound canalso be made in an analogous manner to that of Example 1 above exceptusing 4-chlorophenol in place of 3,4-dichlorophenol,2,5-dichloronitrobenzene in place of 4-chloro-3-nitrobenzotrifluoride,and 2,5-dichlorophenylisocyanate in place of4-chloro-3-trifluoromethylphenyl isocyanate.

EXAMPLE 112-[4-Chloro-2-[3-(6-chloro-α,α,α-trifluoro-3-tolyl)ureido)phenyloxy]-4,5-dichlorobenzenesulfonicacid

The title compound was purchased commercially and is also referred to as4,5-Dichloro-2-[4-chloro-2-[[[[2-chloro-5-(trifluoromethyl)phenyl]-amino]carbonyl]amino]phenoxy]-benzenesulfonicacid)

The title compound can also be made in an analogous manner to that ofExample 1 above except using 2,5 dichloronitrobenzene in place of4-chloro-3-nitrobenzotrifluoride and 2-chloro-5-trifluoromethylphenylisocyanate in place of 4-chloro-3-trifluoromethylphenyl isocyanate.

EXAMPLE 125-Chloro-2-[(4-chloro-2-[3-(4-chloro-α,α,α-trifluoro-3-tolyl)ureido)phenoxy]-4-toluenebenzenesulfonicacid

The title compound was purchased commercially and is also referred to as5-Chloro-2-[4-chloro-2-[[[[4-chloro-3-(trifluoromethyl)phenyl]amino]carbonyl]amino]-phenoxy]-4-methylbenzenesulfonicacid.

The title compound can also be made in an analogous manner to that ofExample 1 above except using 4-chloro-3-methylphenol in place of3,4-dichlorophenol, and 2,5-dichloronitrobenzene in place of4-chloro-3-nitrobenzotrifluoride.

EXAMPLE 135-Chloro-2-[(4-chloro-2-[3-(3,4-dichlorophenyl)ureido)phenyloxy]-4-toluenesulfonicacid

The title compound was purchased commercially. The title compound canalso be made in an analogous manner to that of Example 1 above exceptusing 4-chloro-3-methylphenol in place of 3,4-dichlorophenol,2,5-dichloronitrobenzene in place of 4-chloro-3-nitrobenzotrifluoride,and 3,4-dichlorophenyl isocyanate in place of4-chloro-3-trifluoromethylphenyl isocyanate.

EXAMPLE 145-(1,1-Dimethylpropyl)-[2-[2-[3-(α,α,α-trifluoro-3tolyl)ureido]-α,α,α-trifluoro-4-olyloxy)benzene-sulfonicacid

The title compound is also referred to as5-Butyl-2-[4-(trifluoromethyl)-2-[[[[3-(trifluoromethyl)phenyl]amino]carbonyl]amino]phenoxy]-benzenesulfonicacid and was mande in an analagous manner to that of Example 2 and 3above. MS (FAB) m/e 591 [M+H]⁺.

The following compounds were all purchased commercially. The compounds,however, can also be made by one of skill in the art in an analogousmanner to the Examples illustrated above.

EXAMPLE 152-[2-[3-(4-Chloro-2-tolyl)ureido]-4-chlorophenoxy]-3,5-dichlorobenzenesulfonicacid EXAMPLE 165-Chloro-2-[4-chloro-2-[3-(α,α,α,α',α',.alpha.'-hexafluoro-3,5-xylyl)ureido)phenoxy]-benzenesulfonicacid; also referred to as2-[2-[[[[3,5-Bis(trifluoromethyl)-phenyl]amino]carbonyl]amino]-4-chlorophenoxy]-5-chlorobenzenesulfonicacid EXAMPLE 174,5-Dichloro-2-[4-chloro-2-(3-(4-chloro-α,α,α-trifluoro-3-tolyl)ureido)phenoxy]benzene-sulfonicacid; also referred to as4,5-Dichloro-2-[4-chloro-2-[[[[4-chloro-3-(trifluoromethyl)phenyl]amino]carbonyl]amino]phenoxy]benzenesulfonicacid EXAMPLE 185-Chloro-2-[4-chloro-2-(3-(6-chloro-α,α,α-trifluoro-3-tolyl)ureido)phenoxy]benzene-sulfonicacid; also referred to as 5-Chloro-2-[4-chloro-2-[[[[2-chloro-5-(trifluoromethyl)phenyl]amino]carbonyl]amino]phenoxy]benzenesulfonicacid EXAMPLE 195-Chloro-2-[4-chloro-2-(3-(4-chloro-α,α,α-trifluoro-3-tolyl)ureido)phenoxy]benzene-sulfonicacid; also referred to as5-Chloro-2-[4-chloro-2-[[[[4-chloro-3-(trifluoromethyl)phenyl]amino]carbonyl]amino]phenoxy]benzenesulfonicacid EXAMPLE 205-Chloro-2-(4-chloro-2-(3,4-dichlorophenylaminocarbonylaminophenoxy)benzenesulfonic acid EXAMPLE 215-Chloro-2-(4-chloro-2-(2,3-dichlorophenylaminocarbonylaminophenoxy)benzenesulfonic acid EXAMPLE 225-Chloro-2-(4-chloro-2-(4-chlorophenylaminocarbonylaminophenoxy)benzenesulfonic acid EXAMPLE 235-Chloro-2-(4-chloro-2-(3-trifluoromethylphenylaminocarbonylaminophenoxy)benzenesulfonic acid EXAMPLE 245-Chloro-2-(4-chloro-2-(3-chloro-4-(4-chlorophenoxy)phenylaminocarbonylamino)-phenyoxybenzenesulfonic acid EXAMPLE 255-Chloro-2-(4-chloro-2-(2-(3-chlorophenoxy)-5-trifluoromethylphenylaminocarbonylamino)phenyoxybenezenesulfonic acid EXAMPLE 265-Chloro-2-[4-chloro-2-(5-chloro-2-(4-chlorophenoxy)phenylaminocarbonylamino)phenyoxy]benzenesulfonic acid. METHODS OF TREATMENT

The compounds of Formula (I) and/or Formula (II) or pharmaceuticallyacceptable salts thereof can be used in the manufacture of a medicamentfor the prophylactic or therapeutic treatment of an inflammatory diseasestate in a mammal, preferably a human.

Inhibition of PLA₂ and/or CoA-IT and the simultaneous reduction of PAF,free arachidonic acid and eicosanoid release: from inflammatory cellsaccording to this invention is of therapeutic benefit in a broad rangeof diseases or disorders. The invention herein is therefore useful totreat such disease states both in humans and in other mammals.

Inhibition of CoA-IT and 14 kDa PLA₂ by the compounds of Formula (I)and/or Formula (II) is an effective means for simultaneously reducingPAF, free arachidonic acid and eicosanoids produced in inflammatorycells. The therapeutic utility of blocking lipid mediator generation hasbeen recognized for many years. For example, inhibitors ofcyclooxygenase, such as aspirin, indomethacin, acetaminophen andibuprofen, have demonstrated broad therapeutic utilities. CoA-ITinhibitors inhibit cyclooxygenase products. Another class of inhibitorswhich are used in a broad range of inflammatory disorders are thecorticosteroids. Corticosteroids act in a variety of ways, e.g. toinduce inflammatory cells to produce proteins which inhibit freearachidonic acid release or to down regulate PLA₂ mRNA formation. Both14 kDa PLA₂ inhibitors and CoA-IT inhibitors block the release of freearachidonic acid. Inhibitors of 5-lipoxygenase block the production ofleukotrienes and leukotriene antagonists prevent the bioactions ofleukotrienes. Recent studies indicate that both will have broadtherapeutic utilities. Both 14 kDa PLA₂ inhibitors and CoA-IT inhibitorsblock the production of leukotrienes. Inhibitors of phospholipase A₂block the release of free arachidonic acid and the formation of lyso PAF(the immediate precursor of PAF). PLA₂ inhibitors are recognized to havebroad therapeutic utilities. It does not, however, follow that thedisease states noted above must in fact be caused by altered CoA-IT orPLA₂ activity. Thus, the disease state itself may not be directlymediated by CoA-IT or PLA₂ activity. It only follows that CoA-IT or PLA₂activity is required for the continued expression of symptoms of thedisease state and that CoA-IT or PLA₂ inhibitors will be beneficialagainst the symptoms of these disease states.

Recognition that 14 kDa PLA₂ and/or CoA-IT inhibitors reduce PAFproduction has a number of therapeutic implications. PAF itself has beenimplicated as being involved in a number of medical conditions. Thus incirculatory shock, which is characterised by systemic hypotension,pulmonary hypertension and increased lung vascular permeability, thesymptoms can be mimicked by infusion of PAF. This coupled with evidenceshowing that circulating PAF levels are increased by endotoxin infusionindicate that PAF is a prime mediator in certain forms of shock.

Intravenous infusion of PAF at doses of 20-200 pmol kg<-1>min<-1>intorats has been reported to result in the formation of extensivehaemorrhagic erosions in the gastric mucosa. Thus PAF is the most potentgastric ulcerogen yet described whose endogenous release may underlie orcontribute to certain forms of gastric ulceration. Psoriasis is aninflammatory and proliferative disease characterised by skin lesions.PAF is proinflammatory and has been isolated from lesioned scale ofpsoriatic patients indicating PAF has a role is the disease ofpsoriasis. And finally, increasing evidence supports a potentialpatho-physiological role for PAF in cardiovascular disease. Thus recentstudies in angina patients show PAF is released during atrial pacing.Intracoronary injection of PAF in pigs induces a prolonged decrease incoronary flow and, in guinea pig hearts, it induces regional shuntingand ischaemia. In addition PAF has been shown to initiate thrombusformation in a mesenteric artery preparation, both w:hen administeredexogenously and when released endogenously. More recently PAF has beenshown to play a role in brain ischaemia induced in animal models ofstroke. Thus the compounds of the invention, by virtue of their abilityto antagonise either CoA-IT and/or PLA₂, thus block the production ofPAF, free arachidonic acid and its metabolites, are likely to be ofvalue in the treatment of any of the above conditions.

The action of a PLA₂ inhibitor can be distinguished from the activity ofa CoA-IT inhibitor based on their specific actions on their respectiveenzymes and by their different effects in cellular assays. For exampleonly CoAL-IT inhibitors have the ability to interfere with themobilization of radiolabelled arachidonic acid to move from the alkyl-PCpool to the alkenyl PE pool. Selective inhibitors of 14 kDa PLA₂ arewithout an effect in this assay (assay E). Alternatively, CoA-ITinhibitors will inhibit both LTC₄ and PGE₂ release from activatedmonocytes while selective PLA₂ inhibitors inhibit LTC4 release but spareprostanoid formation or production (assay F).

Disease states which could benefit from the inhibition of lipid mediatorproduction include, but are not limited to, adult respiratory distresssyndrome, asthma, arthritis, reperfusion injury, endotoxic shock,inflammatory bowel disease, allergic rhinitis and various inflammatoryskin disorders. Each of these disorders is mediated in some part bylipid mediators of inflammation. Compounds which inhibit CoA-IT, byvirtue of their ability to block the generation of lipid mediators ofinflammation, are of value in the treatment of any of these conditions.Similarly compounds which inhibit PLA₂, by virtue of their ability toblock the generation of lipid mediators of inflammation stemming fromactivation and/or release of this enzyme are of value in the treatmentof these conditions. In particular, an inhibitor of CoAIT, for instancewould offer an advantage over the classical NSAIDs which affect onlyprostanoid production (and not PAF biosynthesis) thereby inhibiting boththe acute and cell-mediated "chronic" inflammatory processes. Further anadvantage of the PLA₂ inhibitor would be their affect on human monocyteleukotrienes and PAF formation, while immunosuppressive prostanoids,such as PGE₂, are spared.

Treatment of disease states caused by these lipid inflammatory mediatorsi.e., arachidonate, eicosanoids and PAF, include certain cardiovasculardisorders such as but not limited to, myocardial infarction, stroke,circulatory shock, or hypotension, ischemia, reperfusion injury,inflammatory diseases such as, but not limited to, arthritis,inflammatory bowel disease, Crohn's disease, or ulcerative colitis,respiratory disease such as but not limited to, asthma, or adultrespiratory distress syndrome, analphylaxis, shock such as but notlimited to endotoxic shock, topical disesases, such as but not limitedto actinic keratosis; psoriasis, or contact dermatitis, or pyresis.

In order to use a compound of formula (I) or (II) or a pharmaceuticallyacceptable salt thereof in therapy, it will normally be formulated intoa pharmaceutical composition in accordance with standard pharmaceuticalpractice. This invention, therefore, also relates to a pharmaceuticalcomposition comprising an effective, non-toxic mount of a compound offormula (I) or (ID and a pharmaceutically acceptable cattier or diluent.

Compounds of formula (I) or (II), pharmaceutically acceptable saltsthereof and pharmaceutical compositions incorporating such mayconveniently be administered by any of the routes conventionally usedfor drug administration, for instance, orally, topically, parenterallyor by inhalation. The compounds of formula (I) or (II) may beadministered in conventional dosage forms prepared by combining acompound of formula (I) or (II) with standard pharmaceutical carriersaccording to conventional procedures. Such pharmaceutically acceptablecarriers or diluents and methods of making are well known to those ofskill in the art, and reference can be found in such texts asRemington's Pharmaceutical Sciences, 18th Ed., Alfonso R. Genarao, Ed.,1990, Mack Publishing Co. and the Handbook of Pharmaceutical Excipents,APhA Publications, 1986.

The compounds of formula (I) or (II) may also be administered inconventional dosages in combination with known second therapeuticallyactive compounds, such as steroids or NSAID's for instance. Theseprocedures may involve mixing, granulating and compressing or dissolvingthe ingredients as appropriate to the desired preparation. It will beappreciated that the form and character of the pharmaceuticallyacceptable carrier or diluent is dictated by the amount of activeingredient with which it is to be combined, the route of administrationand other well-known variables. The carrier(s) must be "acceptable" inthe sense of being compatible with the other ingredients of theformulation and not deleterious to the recipient thereof.

The pharmaceutical carder employed may be, for example, either a solidor liquid. Exemplary of solid carriers are lactose, terra alba, sucrose,talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acidand the like. Exemplary of liquid carriers are syrup, peanut oil, oliveoil, water and the like. Similarly, the carrier or diluent may includetime delay material well known to the art, such as glycerylmono-stearate or glyceryl distearate alone or with a wax.

A wide variety of pharmaceutical forms can be employed. Thus, if a solidcarrier is used, the preparation can be tableted, placed in a hardgelatin capsule in powder or pellet form or in the form of a troche orlozenge. The amount of solid carrier will vary widely but preferablywill be from about 25 mg. to about 1 g. When a liquid carrier is used,the preparation will be in the form of a syrup, emulsion, soft gelatincapsule, sterile injectable liquid such as an ampule or nonaqueousliquid suspension.

Compounds of formula (I) or (II) may be administered topically, that isby non-systemic administration. This includes the application of acompound of formula (I) or (II) externally to the epidermis or thebuccal cavity and the instigation of such a compound into the ear, eyeand nose, such that the compound does not significantly enter the bloodstream. In contrast, systemic administration refers to oral,intravenous, intraperitoneal and intramuscular administration.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin tothe site of inflammation such as liniments, lotions, creams, ointmentsor pastes, and drops suitable for administration to the eye, ear ornose. The active ingredient may comprise, for topical administration,from 0.001% to 10% w/w, for instance from 1% to 2% by weight of theformulation. It may however comprise as much as 10% w/w but preferablywill comprise less than 5% w/w, more preferably from 0.1% to 1% w/w ofthe formulation.

Lotions according to the present invention include those suitable forapplication to the skin or eye. An eye lotion may comprise a sterileaqueous solution optionally containing a bactericide and may be preparedby methods similar to those for the preparation of drops. Lotions orliniments for application to the skin may also include an agent tohasten drying and to cool the skin, such as an alcohol or acetone,and/or a moisturizer such as glycerol or an oil such as castor oil orarachis oil.

Creams, ointments or pastes according to the present invention aresemi-solid formulations of the active ingredient for externalapplication. They may be made by mixing the active ingredient infinely-divided or powdered form, alone or in solution or suspension inan aqueous or non-aqueous fluid, with the aid of suitable machinery,with a greasy or non-greasy base. The base may comprise hydrocarbonssuch as hard, soft or liquid paraffin, glycerol, beeswax, a metallicsoap; a mucilage; an off of natural origin such as almond, corn,arachis, castor or olive oil; wool fat or its derivatives or a fattyacid such as steric or oleic acid together with an alcohol such aspropylene glycol or a macrogel. The formulation may incorporate anysuitable surface active agent such as an anionic, cationic or non-ionicsurfactant such as a sorbitan esteror a polyoxyethylene derivativethereof. Suspending agents such as natural gums, cellulose derivativesor inorganic materials such as silicaceous silicas, and otheringredients such as lanolin, may also be included.

Drops according to the present invention may comprise sterile aqueous oroily solutions or suspensions and may be prepared by dissolving theactive ingredient in a suitable aqueous solution of a bactericidaland/or fungicidal agent and/or any other suitable preservative, andpreferably including a surface active agent. The resulting solution maythen be clarified by filtration, transferred to .a suitable containerwhich is then sealed and sterilized by autoclaving or maintaining at98°-100° C. for hag an hour. Alternatively, the solution may besterilized by filtration and transferred to the container by an aseptictechnique. Examples of bactericidal and fungicidal agents suitable forinclusion in the drops are phenylmercuric nitrate or acetate (0.002%),benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%).Suitable solvents for the preparation of an oily solution includeglycerol, diluted alcohol and propylene glycol.

Each dosage unit for oral administration contains preferably from 1 to250 mg (and for parenteral administration contains preferably from 0.1to 25 mg) of a compound of the structure (I) or a pharmaceuticallyacceptable salt thereof calculated as the free base.

The pharmaceutically acceptable compounds of the invention will normallybe administered to a subject in a dally dosage regimen. For an adultpatient this may be, for example, an oral dose of between 1 mg and 500mg, preferably between 1 mg and 250 mg, or an intravenous, subcutaneous,or intramuscular dose of between 0.1 mg and 100 mg, preferably between0.1 mg and 25 mg, of the compound of the Formula (I)/(II) or apharmaceutically acceptable salt thereof calculated as the free base,the compound being administered from 1 to 4 times per day.

The choice of form for administration, as well as effective dosages,will vary depending, inter alia, on the condition being treated. Thechoice of mode of administration and dosage is within the skill of theart.

BIOLOGICAL METHODS

To determine activity of the compounds of Formula (I) and (II) variouscellular assays can be used to determine in vitro activity.Additionally, various classical in vivo acute inflammatory models whichhave some aspect of their etilogy to elevated eicosanoid levels can beemployed, such as the paw edema model, mouse zymosan peritonitis,reverse Arthus pleurisy or various skin inflammation assays which aredescribed in Lewis et al., Experimental Models of inflammation, in theHandbook of Inflammation, Vol. 5, Bonta Ed., Elsevier SciencePublishers, New York (1985) whose disclosure is herein incorporated byreference. The TPA induced ear edema model (mouse) as well as thecarrageenan paw edema model in the rat are described herein as well.These classical models of inflammation will reflect the drug's abilityto alter an inflammatory response but cannot address the specificity ofdrug action. These models have been traditionally designed as nonsteriod antiinflammatory drug sensitive pharmacological screens and itis important to utilize models which can differentiate PLA2 and CoA-ITinhibitors from NSAIDS.

Cell-free and Cellular Assessment of Inhibitors

Described herein are several in vitro assays both for CoA-IT and PLA₂enzyme activities. The first employes purified recombinant enzyme or abroken cell assay, assay (a or b, respectively) described below.Alternatively, evaluation of inhibitors can occur in intact cells suchas described in the assay, assay (c and d) below. CoA-IT activity canexclusively be measured, and differentiated from PLA₂ inhibition, inintact cells by following the movement of a pulse of [³ H] arachidonateas it moves into the 1-alkyl and 1-alkenyl phospholipids in inflammatorycells (assay e). It should be noted for the purposes herein that assaysc, d, & f can both be used for PLA₂ and CoA-IT inhibition determination.

Inflammatory Responses in vivo

The ability of compounds that inhibit CoA-IT and/or PLA₂ to affect invivo inflammatory responses may be assessed. Inflammatory responses areinduced in the mouse ear by the topical application of apro-inflammatory agent, such as 12-0-tetradecanoylphorbol 13-acetate(assay g). This produces an edematous response, as measured by increasesin ear thickness, as well as increased inflammatory cellular infiltrate,as measured by increases in myeloperoxidase activity (as described inthe methods). To further validate the mechanism of action inflammationinduced by the direct administration of arachidonic acid can be used. Inthis case, compounds altering arachidonic acid mobilization orliberation should be without effect.

IN VITRO ASSAYS

Assay (a): Phospholipase A₂ assay:

Phospholipase A₂ activity of rh Type II-14 KDaPLA₂ or PLA₂ semi-purifiedfrom human Synovial joint fluid was measured by the acylhydrolysis ofhigh specific activity (NEN)[³ H]-AA-E. coli (0.5 mCi/5 nmol PL Pi) aspreviously described in Marshall et al., J. Rheumatology, 18:1, pp59-65(1991). High specific activity [³ H]AA-E. coli had up to 95% of thelabel incorporated into phospholipid which was localized almostexclusively in the sn-2 position, as demonstrated by purified 14 kDaPLA₂ or low molecular weight PLA₂ acylhydrolysis and separation ofproducts by thin layer chromatography (TLC) (data not shown).[Predominately used herein was rh Type II 14 kDa PLA2, or alternativelybovine pancreatic PLA₂ was also be used]. The reaction mixture (50 or100 ml total volume) contained 25 mM HEPES, pH 7.4, 150 mM NaCl, 5 mMCaCl₂ and [³ H]-AA-E. coli (low specific activity; 5-8 nmol PL Pi perassay). Assays were incubated for a time predetermined to be on thelinear portion of a time versus hydrolysis plot (10 min). Experimentswere conducted with final % hydrolysis values ranging from 2% (400-1000dpm) to 10% (2000-5000 dpm) acylhydrolysis after blank correction.Reactions were terminated by the addition of 1.0 mL tetrahydrofuran(THF). The whole sample was placed over aminopropyl solid phase silicacolumns and eluted with THF:acetic acid (49:1 ) exclusively separatingfree fatty acids with greater than 95% recovery. Radiolabel in thiseluate was quantitated by liquid scintillation counting. Results wereexpressed as % of fatty acid hydrolyzed ([sample dpms-non-specific(blank) dpms/total dpms]×100) or specific activity which was calculatedfrom hydrolysis values found in the linear portion of time versus %hydrolysis plots (pmol free fatty acid hydrolyzed/mg/min). Non-specificactivity was always less than 1% of the total counts added.

Protein determination

All protein concentrations were determined by Bradford protein analysiskits (Biorad, Richmond, Calif.).

Results:

The following representative compounds of Formula (I) and (II) belowdemonstrated positive PLA₂ inhibition in the above noted method. Whilethese compounds generally tested positive at 50 μm levels, several werealso tested for positive inhibitory activity at up to 500 μM levels.Such compounds include:

5-Chloro-2-[(4-chloro-2-(3-(2,5-dichlorophenyl)ureido)phenyloxy]benzenesulfonicacid;

2-[4-Chloro-2-[3-(6-chloro-α,α,α-trifluoro-3-tolyl)ureido)phenyloxy]-4,5-dichlorobenzenesulfonicacid;

5-Chloro-2-[(4-chloro-2-[3-(3,4-dichlorophenyl)ureido)phenyloxy]-4-toluenesulfonicacid;

2-[2-[3-(4-Chloro-2-tolyl)ureido]-4-chlorophenoxy]-3,5-dichlorobenzenesulfonicacid;

2-[2-[3-(4-Chloro-3-trifluoromethylphenyl)ureido]-4-trifluoromethylphenoxy]-4,5-dichlorobenzenesulfonic acid;

5-Chloro-2-[4-chloro-2-[3-(α,α,α,α',α',.alpha.'-hexafluoro-3,5-xylyl)ureido)phenoxy]-benzenesulfonicacid;

5-(1,1-Dimethylpropyl)-[2-[2-[3-(α,α,α-trifluoro-3-tolyl)ureido]-α,α,α-trifluoro-4-tolyloxy)benzenesulfonicacid;

2-[2-[3-(4-Chloro-3-trifluoromethylphenyl)ureido]-4-trifluoromethylphenoxy]-5-(1,1-dimethethylpropyl)benzenesulfonicacid;

2-[[2-[3-(α,α,α,α',α',α'-hexafluoro-3,5-xylyl)ureido]-α,α,α-trifluroro-4-tolyloxy]-5-(1,1-dimethylpropyl)benzenesulfonicacid;

5-Chloro-2-[4-chloro-2-(3-(4-chloro-α,α,α-trifluoro-3-tyl)ureido)phenoxy]benzenesulfonicacid;

5-Chloro-2-(4-chloro-2-(3,4-dichlorophenylaminocarbonylaminophenoxy)benzenesulfonic acid;

5-Chloro-2-(4-chloro-2-(2,3-dichlorophenylaminocarbonylaminophenoxy)benzenesulfonic acid;

5-Chloro-2-(4-chloro-2-(4-chlorophenylaminocarbonylaminophenoxy)benzenesulfonic acid;

5-Chloro-2-(4-chloro-2-(3-trifluoromethylphenylaminocarbonylaminophenoxy)benzenesulfonic acid;

5-Chloro-2-[4-chloro-2-(2-chloro-5-trifluoromethylphenylamino)carbonylamino]phenoxybenzenesulfonic acid;

5-Chloro-2-(4-chloro-2-(3-chloro-4-(4-chlorophenoxy)-phenylaminocarbonylamino)phenyoxybenzenesulfonic acid;

5-Chloro-2-(4-chloro-2-(2-(3-chlorophenoxy)-5-trifluoromethylphenylaminocarbonylamino)phenyoxybenzenesulfonic acid; and

5-Chloro-2-[4-chloro-2-(5-chloro-2-(4-chlorophenoxy)phenylaminocarbonylamino)phenyoxy]benzenesulfonic acid.

Assay (b): CoA-IT Activity

The following is a method to measure CoA-IT activity and the effects ofcompounds on CoA-IT activity. The assay is based upon mixing cellularmaterial containing CoA-IT activity with a stable lyso phospholipid suchas 1-alkyl-2-acyl-GPC and measuring the production of phospholipidproduct such as 1-alkyl-2-acyl-GPC occurring in the absence of added CoAor CoA-fatty acids.

Cell Preparation

Any inflammatory cell that contains high levels of CoA-IT activity canbe used, such as neutrophils, macrophages or cell lines such as U937cells. U937 cells were obtained from American Type Culture Collectionand grown in RPMI-1640 media (Gibco, Grand Island, N.Y.) supplementedwith 10% fetal bovine serum (Hyclone, Logan, Utah) at 37° C., 5% C0₂.Cells were grown without differentiation (basal state) by any agent,such as dimethyl sulfoxide. As used herein, "inflammatory cells"include, but are not limited to neutrophils, macrophages, monocytes,lymphocytes, eosinophils, basophils, and mast cells.

Microsomal preparation

Microsomes were prepared using standard techniques. In this case, cellswere washed with a buffer of 250 mM sucrose, 10 mM Tris, 1 mM EGTA, 1 mMMgCl₂, pH 7.4 and ruptured by N₂ cavitation (750 psi, 10 minutes). Theruptured cells were centrifuged 1000×g, 5 minutes. The resultingsupernatant was centrifuged at 20,000×g,˜20 minutes. Microsomes wereprepared from this supernatant by centrifugation at 100,000×g, 60minutes. The resulting pellet was washed once with assay buffer (150 mMNaCl, 10 mM Na₂ KPO₄, 1 mM EGTA, pH 7.4), recentrifuged and the pelletresuspended in assay buffer (4-20 mg protein/ml) and was stored at -80°C. until assayed.

CoA-IT activity

CoA-IT activity was measured in 1.5 ml centrifuge tubes in a totalvolume of 100 ul. Microsomes were diluted in assay buffer to the desiredprotein concentration (6-20 ug/tube). The reaction was initiated byaddition of [3H ]1-alkyl-2-lyso-sn-glycero-3-phosphocholine (GPC) (˜0.1uCi/tube) and 1 μM final cold 1-alkyl-2-lyso-GPC in assay buffer with0.25 mg/ml fatty acid-poor bovine serumalbumin (BSA) (Calbiochem, LaJolla, Calif.). [³ H]1-alkyl-2-lyso-GPC, approximately 50 Ci/mmol, wasfrom NEN-Dupont (Boston, Mass.) and cold 1-alkyl-2-lyso-GPC was fromBiomol (Plymouth Meeting, Penn.). Microsomes were pretreated withdesired agents for the desired time (10 minutes) before the addition of[³ H]1-alkyl-2-lyso-GPC. The reaction was run for the desired time (10minutes) at 37° C. The reaction was stopped and the lipids extracted byaddition of 100 ul of chloroform:methanol (1:2, v/v) followed by 100 ulof chloroform and 100 ul of 1M KCI. The samples were vortexed andcentrifuged at high speed in a microfuge for 2-3 minutes. An aliquot ofthe chloroform-extracted materials were separated, usually by TLC inchloroform/methanol/acetic acid/water (50:25:8:4, v/V), visualized byradioscanning (Bioscan) and the product, [³ H]1-alkyl-2-acyl-GPC, wasscraped and quantified by liquid scintillation spectroscopy. With thisTLC system, the synthetic standards of 1-alkyl-2-lyso-GPC and1-alkyl-2-acyl-GPC were well separated, with Rf values of approximately0.25 and 0.65, respectively. Other methods can be used to separatesubstrate from product, including but not limited to columnchromatography, affinity chromatography and post reactionderivitization.

Protein concentration were assessed using the protein assay reagentsfrom Bio-Rad (Richmond, Calif.).

Result

A variety of compounds have been tested in this assay to determine itsselectivity and inability to detect trivial, non-selective inhibitors.Inhibitors of 5-lipoxygenase (5-LO) and cyclooxygenase (CO), such asindomethicin, naproxen,6-(4'-Fluorophenyl)-5-(4-pyridyl)-2,3-dihydroimidzo-[2,1-b]thiazole and6-(4'-Fluorophenyl)-5-(4-pyridyl)2,3-dihydroimidzo-[2,1-b]thiazole-dioxidehad no effect in CoA-IT activity at concentrations up to 100 μM. Theanti-oxidant BHT also has no effect at concentrations up to 100 μM.Compounds which complex with phospholipids and inhibit PLA₂ activity,such as quinacrine and aristolochic acid have no effect on CoA-ITactivity at concentrations up to 500 μM. Doxepine, a compound reportedto inhibit PAF release did not inhibit CoA-IT at concentrations up to100 μM. Sodium diclofenac, reported to decrease leukotriene productionby altering arachidonic acid metabolism, had no effect on CoA-ITactivity at concentrations up to 500 μM. These results show that theassay for CoA-IT activity is sensitive and selective.

Representative compounds of Formula (I) and (II) which inhibit CoA-ITactivity in the microsomal CoA-IT assay above [generally at 50 μM orless] are:

4,5-Dichloro-2-[4-trifluoromethyl-2-(3-trifluoromethyl-4-chlorophenyl)ureido)phenyl)thio]benzenesulfonicacid;

2-[2-[3-(4-Chloro-3-(trifluoromethyl)phenyl]ureido]-4-(trifluoromethyl)phenoxy]-4,5-dichlorobenzenesulfonicacid, sodium salt;

2-[2-[3-[3,5-Bis(trifluoromethyl)phenyl]ureido]-4-trifluoromethylphenoxy]-5-(1,1-dimethylpropyl)benzenesulfonicacid, sodium salt;

5-Chloro-2-[(4-chloro-2-[3-(4-chloro-α,α,α-trifluoro-3-tolyl)ureido)phenoxy]-4-toluene-benzenesulfonicacid;

4,5-Dichloro-2-[4-chloro-2-(3-(4-chloro-α,α,α-trifluoro-3-tolyl)ureido)phenoxy]benzene-sulfonicacid; and

5-Chloro-2-(4-chloro-2-(3,4-dichlorophenylaminocarbonylaminophenoxy)benzenesulfonic acid.

Assay (c): Arachidonic Acid Release Assay

Preparation of human neutrophils

Human neutrophils are obtained in the laboratory using three differentmethods. One method uses leukophoresis packs from normal humans andneutrophils are isolated using the histopaque-1077 technique. The bloodis centrifuged at 300×g for 10 minutes. The cell pellets are resuspendedin PBS composed of 137 mM NaCI, 8.8 mM Na2HPO4, 1.5 mM KH2PO4, 2.7 mMKCI (Dulbecco's Gibco Laboratories, Long Island, N.Y.) and layered overhistopaque-1077 (Sigma, St. Louis, Mo.). The pellets are collected aftercentrifugation (300×g for 30 minutes) and washed once in PBS. The cellpellets are exposed briefly to deionized water to lyse any erythrocytes.The remaining cells are collected by centrifugation, suspended in PBS,counted and identified after cytospinning and staining. The finalleukocyte preparation will be of greater than 95% purity and viability.

The second method isolates human neutrophils from fresh heparinizednormal blood using the Histopaque-1077 technique. The blood is layeredover Histopaque-1077 (Sigma, St. Louis Mo.) and centrifuged at 400×g for30 minutes. The cell pellets are resuspended in 35 ml of PBS and 12 mlof 6% Dextran, followed by Dextran sedimentation at room temperature for45 minutes. The upper layer is collected and further centrifugated for10 minutes at 1000 rpm. The cell pellets are exposed briefly todeionized water to lyse erythrocytes. The remaining cells are collectedby centrifugation, suspended in PBS, counted and identified aftercytospinning and staining. The final leukocyte preparation will be ofgreater than 95% purity and viability.

The third method isolates human neutrophils from freshly drawnheparinized normal blood using the Percoll technique. The, blood isfirst treated with 6% Dextran at room temperature for a 1 hoursedmination. The upper layers of plasma are collected and centrifuged at400×g for 10 minutes. The cell pellets are resuspended in Percoll 1.070g/ml supplemented with 5% fetal bovine serum and layered ondiscontinuous gradients (1.080, 1.085, 1.090,1.095 g/ml) followed bycentrifugation at 400×g for 45 minutes. The neutrophils are collectedfrom interfaces of 1;080 and 1.085 and the 1.085 and 1.090 Percolldensities, followed by a centrifugation at 400×g for 45 minutes. Theneutrophils are suspended in PBS, counted and identified aftercytospinning and staining. The final leukocyte preparation will be ofgreater than 95% purity and viability.

There should be no difference noted in the response of the neutrophilsnor in the effects of test compounds in neutrophils isolated by thethree different techniques.

Treatment of human neutrophils

Neutrophils are suspended in PBS with 1 mM Ca²⁺ and 1.1 mM Mg²⁺ atconcentrations of 5 to 20×106 cells per ml. Cells are added to testtubes and treated with the desired compounds for 5 to 10 minutes, thenchallenged with calcium ionophere A23187, 2 μM, or vehicle control, PBScontaining 0.25-1 mg/ml BSA. After 5 to 20 minutes, the reactions areterminated by addition of an equal volume of chloroform:methanol (1:2,v/v) to the samples. [² H₈ ]Arachidonic acid (50, 100 or 200 ng) isadded as an internal standard and the lipids ware extracted by additionof equal volumes of chloroform and distilled water. The samples arevortexed and centrifuged at high speed and the chloroform layer removedto a clean tube.

Assay for free arachidonic acid

The chloroform extract for each sample is evaporated to dryness and thematerial resuspended in hexane. The hexane is passed through a Silicasolid phase column (500 mg), washed 2× with hexane and a fatty acidenriched fraction eluted with hexane:ethyl ether (1:1, v/v). Solventsare removed from the samples under a stream of nitrogen then the samplesare converted to pentafluorobenzyl esters using pentafluorobenzylbromide and diisopropylethylamine in acetronitrile. Solvents are removedand samples are suspended in hexane. GC/MS analysis is performed on asuitable instrument, such as a Finnigan MAT TSQ 700 GC/MS/MS/DS (SanJose, Calif.) operated as a single stage quadruple system or aHewlett-Packard 5890 with a 5989A M5 system.

The peaks corresponding to arachidonic acid and [² H₈ ]Arachidonic acidare identified and the areas of those peaks compared and the releasedarachidonic acid calculated as ng of arachidonic acid for each sample.

Protein concentrations are assessed using the protein assay reagentsfrom Bio-Rad (Richmond, Calif.).

A representative compound herein which demonstrated positive activity,i.e., inhibition of arachidonic acid release in this assay are:

2-[2-[3-(4-Chloro-3-trifluromethylphenyl)ureido]-4-trifluoromethylphenoxy]-4,5-dichlorobenzenesulfonic acid; and

2-[2-[[[[3,5-Bis(trifluoromethyl)phenyl]amino]carbonyl]amino]-4-(trifluoromethyl)phenoxy]-5-(1,1-dimethylpropyl)benzenesulfonicacid;

Assay (d): Assay for Production of Platelet-Activating Factor (PAF)

Preparation of human neutrophils:

Blood is obtained from normal humans and neutrophils are isolated asdescribed for the arachidonic acid release assay, above. The finalleukocyte preparation should be of greater than 95% purity andviability.

Treatment of human neutrophils

Neutrophils are suspended in PBS at concentrations of 5 to 20×10⁶ cellsper ml. Cells are added to test tubes and treated with the desiredcompounds for 5 to 10 minutes, then challenged with calcium ionophoreA23187, 2 μM and 20-30 μCi of [3H]acetic acid (NEN-Dupont, Boston,Mass.), or the vehicle of PBS with 0.25-1 mg/ml. After 5 to 20 minutes,the reactions are terminated by addition of an equal volume ofchloroform:methanol (1:2, v/v) to the samples and the lipids areextracted by addition of equal volumes of chloroform and distilledwater. The samples are vortexed and centrifuged at high speed and thechloroform layer removed to a clean tube.

Assay for PAF

The chloroform from each tube is evaporated to dryness and the materialsuspended in a small volume of chloroform or chloroform:methanol (25-100μl) and the total material spotted on a Silica TLC plate. The plates aredeveloped in chloroform/methanol/acetic acid/water (50:25:8:4, v/v)visualized by radioscanning (Bioscan) and the product, [³ H]PAF, isscraped and quantified by liquid scintillation spectroscopy. With thisTLC system, the Rf value for a synthetic standard of PAF isapproximately 0.33.

A representative compound herein which demonstrated positive activity,i.e., inhibition of PAF production, in this assay is:

2-[2-[3-(4-Chloro-3-trifluoromethylphenyl)ureido]-4-trifluoromethylphenoxy]-4,5-dichlorobenzenesulfonic acid; and

Assay (e): Methods for the evaluation of CoA-IT inhibitors onmobilization of labeled arachidonic acid in intact cells

Measurement of the effect of CoA-IT inhibitors on the transfer of [³H]arachidonate into 1-ether phospholipids in non-stimulated inflammatorycells can be accomplished by general application of the followingspecific method. Human neutrophils were isolated and resuspended (5×10⁷/ml) in Hanks Balanced Salt Solution (HBSS; Gibco).[5,6,8,9,11,12,14,15-³ H]-Arachidonic acid (100 Ci/mmol; New EnglandNuclear) complexed to 200 μl HBSS containing 0.25 mg/ml HSA was added tothe cell suspension (1 μCi/ml). The cells were incubated with gentleshaking at 37° C. for 5 min. The reaction was terminated by the additionof 40 ml ice-cold HBSS containing HSA (0.25 mg/ml). The cells were thenremoved from the supernatant fluid by centrifugation (225 g, 8 min).Unincorporated [³ H]-arachidonic acid was completely removed by two morewashes of HBSS containing 0.25 mg/ml HSA. The neutrophils wereresuspended in fresh buffer, exposed to various concentrations of aCoA-IT inhibitor or its vehicle and incubated without stimulation for 2hrs. At that time, the tubes containing the cells and buffer wereextracted (Bligh & Dyer [Can. J. Biochem. Physiol. (1959) 37, 911-917])and the phospholipid classes separated and collected by normal phaseHPLC, using a Ultrasphere Silica column (4.6 mm×250 mm; Rainin) elutedwith hexane/2-propanol/ethanol/phosphate buffer (pH 7.4)/acetic acid(490:367:100:30:0.6 v/v) for 5 min at a flow rate of 1 ml/min. Theamount of phosphate buffer in the eluting solvent was increased to 5 %over 10 rain and this solvent composition was maintained until all thephospholipid classes had eluted from the column (30-40 min) (Chilton, F.H. [Methods Enzymol. (1990)187, 157-166]). The phospholipids wereconverted into diradylglycerols by addition of phospholipase C, 20units-40 units of Bacillus cereus phospholipase C (Sigma Type XIID in100 mM Tris HCl buffer (pH 7.4) for 2.5-6 hr, then convened into1,2-diradyl-3-acetylglycerols by incubation with acetic anhydride andpyridine (Chilton, F. H. [Methods Enzymol. (1990)187, 157-166]). Thephospholipid subclasses were separated by TLC in benzene/hexane/ethylether (50:45:4, v/v), located by image analysis (Bioscan) and the amountof radioactivity in each class was determined by zonal scraping andliquid scintillation counting.

A representative compound herein which demonstrated positive activity,i.e., blocking the movement of arachidonic acid into 1-etherphospholipids in this assay is:

2-[2-[3-(4-Chloro-3-trifluoromethylphenyl)ureido]-4-trifluoromethylphenoxy]-4,5-dichlorobenzenesulfonic acid; and

The following is the method for assessing the ability of a compound toalter arachidonate content of cellular phospholipids, which can begeneralized for any desired cell. Specifically, mouse bonemarrow-derived mast cells are removed from culture and provided withexogenous [³ H]arachidonic acid for 30 minutes. The labeled arachidonicacid which had not been incorporated into the cells is then removed bywashing the cells 2 times with an albumin-containing buffer. At thatpoint, the cells are treated with various concentrations of CoA-ITinhibitors and then placed back in culture for 24-48 hours. Thephospholipids are extracted by the method of Bligh and Dyer [Can. J.Biochem. Physiol. (1959) 37, 911-917] and phospholipids separated bynormal phase HPLC by the method of Chilton [Methods Enzymol. (1990)187,157-166]. The radioactive and mole quantities of arachidonate in complexlipids are determined. At this point, cellular lipid extracts aretreated with KOH (0.5M) to remove fatty acids from complex lipids(phospholipids) and the quantities of arachidonate in these extracts canthen be determined by various methods, including gas chromatography andmass spectrometry (Chilton [Methods Enzymol. (1990)187, 157-166]).

A representative compound herein which demonstrated positive activity,i.e., decreasing the arachidonic content, in this assay is:

2-[2-[3-(4-Chloro-3-trifluoromethylphenyl)ureido]-4-trifluoromethylphenoxy]-4,5-dichlorobenzenesulfonic acid; and

Assay (f) for measurement of stimulated eicosanoid release by humanmonocytes.

Human Monocyte Isolation. Leukocyte-rich leukopaks obtained fromBiological Specialties (Lansdale, Pa.) were collected from malevolunteers who were not taking anti-inflammatory drugs. Leukopaks werecentrifuged (90×g for 15 min) twice to remove the platelet-rich plasma.The cell pellet was washed by centrifugation and resuspended in HBSSwithout Ca²⁺ or Mg²⁺. Histopaque 1077 was layered under the cellsuspension and centrifuged at 400×g for 30 min to obtain the buffy coat.The interfacial buffy coat, containing monocytes and lymphocytes, wasremoved and saved. The buffy coat was washed twice with HBSS withoutCa²⁺ or Mg²⁺ by centrifugation. The cell pellet (4-6×10⁸ cells/30 mls)was resuspended in iso-osmotic media (RPMI-1640, 10% heat inactivatedfetal bovine serum (FBS), 0.2 mM L-glutamine, 2.5 mM HEPES) and layeredover an equal volume of 46% Percol mixture (10×PBS/Percol; 9.25/0.75)and 54% iso-osmotic media and centrifuged for 30 min at 1000×g (Marshalland Roshak, Biochem. Cell Biol. 71:331-339, 1993). The monocytepopulation located at the interface of the Percoll gradient was removedand washed twice in HBSS without Ca²⁺ or Mg²⁺. This resulted in agreater than 85-90 % pure monocyte population as assessed bydifferential staining.

Measurement of Stimuli-Induced Eicosanoid Release. Monocytes (5×10⁶ /ml)were incubated as a suspension in serum-free RPMI-1640 medium containingthe vehicle DMSO (<1%) or drug for 30 min at 27° C. after which vehicleor stimuli was added for the indicated time. The stimulating agent issolubilized in DMSO and appropriate vehicle controls were included inall experiments. The amount of stimuli was chosen from the linearportion of a concentration versus product curve usually representing60-80% maximal stimulation over the indicated incubation time at 37° C.(A23187, 1 μM,(15 min). The reaction was terminated by reduction of pHthrough addition of citric acid and centrifugation (10 min, 400×g, 4°C.). Cell viability was monitored before and after experiments usingtrypan blue exclusion. The cell-free media was decanted and stored at-70° C. until analyzed. Prostaglandin E₂ and LTC₄ were directly measuredin cell-free media using enzyme immunoassay (EIA) kits purchased fromCaymen Chemical Co. (Ann Arbor, Mich.). Sample or standard dilutionswere made with appropriate media and analyzed in triplicate. Resultswere obtained by extrapolation from a standard curve prepared in themedia and expressed as pg or ng/ml of sample.

A representative compound herein which demonstrated positive activity inthis assay was:

2-[2-[[[[3,5-Bis(trifluoromethyl)phenyl]amino]carbonyl]amino]-4-(trifluoromethyl)-phenoxy]-5-(1,1-dimethylpropyl)benzenesulfonicacid

For instance, the above noted a compound demonstrated (as an IC₅₀)PGE₂ >10 uM, and for LTC₄ 1 uM.

IN VIVO ASSAYS

Assays (g and h): Assay (Method) for TPA (assay g)or Arachidonic acid(assay h)-induced Inflammation

Animals:

Male Balb/c inbred mice were obtained from Charle River BreedingLaboratories (Kingston, N.Y.). Within a single experiment mice (22-25 g)were age-matched. These in vivo experiments typically involved use of5-6 animals/group.

(g) TPA-induced Mouse Ear Inflammation:

Assay of Ear Edema

TPA (12-0-tetradecanoylphorbol 13-acetate) (Sigma Chemical Company) inacetone (4 mg/20 ml) was applied to the inner and outer surfaces of theleft ear of BALB/c male mice. The thickness of both ears was thenmeasured with a dial micrometer (Mitutoyo, Japan) at both 2 and 4 hoursafter treatment, and the data expressed as the change in thickness (10⁻³cm) between treated and untreated ears. The application of acetone didnot cause an edematous response; therefore, the difference in earthickness represented the response to the TPA. After measuring theedema, the inflammed left ears were removed and stored at -70° C. untilthey were assayed for MPO (myeloperoxidase) activity where appropriate.

Assay of Myeloperoxidase (MPO) in Inflamed Ear Tissue:

On the day of the assay, partially thawed ear tissues were minced andthen homogenized (10% w/v) with a Tissumizer homogenizer (Tekmar Co.) in50 mM phosphate buffer (pH 6) containing 0.5% HTAB. The tissuehomogenates were taken through three cycles of freeze-thaw, followed bybrief sonication (10 sec). The method of Bradley et al. was used withmodifications as described. The appearance of a colored product from theMPO-dependent reaction of o-dianisidine (0.167 mg/ml; Sigma) andhydrogen peroxide (0.0005%; Sigma) was measured spectrophotometricallyat 460 nm. Supernatant MPO activity was quantified kinetically (changein absorbance measured over 3 min, sampled at 15-sec intervals) using aBeckman DU-7 spectrophotometer and a Kinetics Analysis package (BeckmanInstruments, Inc.). One unit of MPO activity is defined as thatdegrading one micromole of peroxide per minute at 25° C.

Statistics;

Statistical analysis was done using Student's "t" test. The ED₅₀ arevalues which cause a 50% inhibition of the inflammatory response and arecalculated by regression analysis of the dose response data.

(h) Arachidonic acid induced ear inflammation assay

Arachidonic acid is dissolved in acetone (1 mg/ear) to the left ear ofBALB/c male mice. The thickness of both ears was measured with aconstant pressure thickness guage 1 hour after treatment and the dataexpressed as the change in thickness between treated and untreated ears.Test compounds or vehicle are given at the time of AA application. Theinflammatory cell infiltration is measured by MPO activity as describedabove in the TPA ear edema assay. After the edema measurements are made,the inflamed ears are removed and assayed for MPO activity.

The anti-inflammatory effect of various standard inhibitors topicallyadministered in the AA and TPA induced mouse car edema model weremeasured for dexamethasone, scalaradial and Wyeth's compound WY 50,295at does of 0.2, 0.1 and 0.3 respectively. The TPA % change in edema was-50 (p<0.001), -46 (p<0.01) and -18 (ns) respectively; for AA the changewas -10 (ns), -11(ns) and -50 (p<0.001). The change in MPO for TPA modelwas -54 (p<0.001), -65 (p<0.001) and -36 (p<0.05) respectively; for AAit was 0 (ns), -33 (ns) and -90 (p<0.001). One hypothesis is that the AAadministration to the car overrides the need for PLA₂ mediatedliberation of substrate for subsequent proinflammatory lipid mediatorgeneration or AA mobilization by CoA-IT. In this case an inhibitor of anAA-metabolizing enzyme should be effective while and inhibitor of PLA₂would be ineffective. As noted above, scalaradial and dexamethasone havelittle or no effect in the AA car model at concentrations which wereeffective in the TPA ear model. This can be contrasted to the activityof the selective 5-LO inhibitor WY 50,295 which strongly inhibitsinflammation in response to AA. The AA car model therefore responds wellto compounds that exhibit 5-LO inhibitory action and appears to beuneffected by putative PLA₂ inhibitors. This model therefore provides aunique mol with which the contribution of the 5-LO inhibition to the invivo anti-inflammatory activity of various compounds can be separatedfrom LMW-PLA₂ inhibition.

The compounds of Formula (II),2-[2-[[[[3,5-Bis(trifluoromethyl)phenyl]amino]carbonyl]amino]-4-(trifluoromethyl)-phenoxy]-5-(1,1-dimethylpropyl)benzenesulfonicacid, and2-[2-[3-(4-Chloro-3-trifluoromethylphenyl)ureido]-4-trifluoromethylphenoxy]-5-(1,1-dimethylpropyl)benzenesulfonicacid; demonstrated a positive inhibition in these animal models.

Specifically in the TPA ear model at 50 mg/ear topically both compoundsdemonstrated roughly equipotent inhibitors of the inflammatory cellinfiltration. The compound2-[2-[3-(4-Chloro-3-trifluoromethylphenyl)ureido]-4-trifluoromethylphenoxy]-5-(1,1-dimethylpropyl)-benzenesulfonicacid demonstrated, for edema, an ED₅₀ of 0.32 mg/ear and for MPO andED₅₀ of 0.36 mg/ear and2-[2-[[[[3,5-Bis(trifluoromethyl)phenyl]amino]-carbonyl]amino]-4-(trifluoromethyl)-phenoxy]-5-(1,1-dimethylpropyl)benzenesulfonicacid, demonstrated, for edema, 0.87 mg/ear and for MPO 0.25 mg/ear.

In addition, for the AA ear model the compound2-[2-[3-(4-Chloro-3-trifluoromethylphenyl)ureido]-4-trifluoromethylphenoxy]-5-(1,1-dimethylpropyl)-benzenesulfonicacid demonstrated activity of -22 @ 1 mg and2-[2-[[[[3,5-Bis(trifluoromethyl)phenyl]amino]-carbonyl]amino]-4-(trifluoromethyl)-phenoxy]-5-(1,1-dimethylpropyl)benzenesulfonicacid, demonstrated activity of -25 @ 1 mg in direct contrast to theinformation provided above for Dexamethasone, Scalaradial and the Wyethcompound.

Thus this demonstrates a clear utility in the treatment of topicallyadministered diseases associated with inflammation as noted herein suchas, but not limited to, inflammatory bowel disease, contact dermatoses,actinic keratosis, psoriasis, or conjuctivitis.

Assay (I): DMPM Assay for LMW-PLA₂

The DMPM assay is used to distinguish specific catalytic inhibitors fromnonspecific agents. Catalysis by the low molecular weight PLA₂ involvesa two step process where the enzyme first binds to the water-lipidinterface and then binds and hydrolyzes a single molecule ofphospholipid at the active site. Binding to the interface is areversible process. The relative rates of interfacial binding andsubstrate hydrolysis determine the kinetics in a particular substratesystem. If a compound affects the surface charge, or the "quality" ofthe interface, inhibition may result as the enzyme will spend less timeat the interface, rather than because hydrolysis is inhibited. DMPM hasbeen shown to bind low molecular weight PLA₂ to its water-lipidinterface very tightly so that the hydrolytic reaction becomescompletely processive relative to the interfacial binding. That is, manyhydrolytic cycles (essentially an infinite number) occur before theenzyme falls off the surface. Because interfacial binding involves afairly large part of the enzyme surface as well, this tight binding isunlikely to be affected by reasonable concentrations (less than 10 mole%) of added inhibitors. Using the DMPM substrate eliminates theartifacts that have been intrinsic to more conventional PLA₂ assayswhere the interfacial binding step is an important factor in thereaction kinetics.

Linear inhibition in the DMPM assay, according to a kinetic modelanalogous to a Dixon plot, implies a specific enzyme interaction butcannot distinguish between competitive and non-competitive inhibition.Results are given as Xi₅₀, or mole percent relative to the lipidsubstrate required for 50 % inhibition.

Preparation of Substrate:

0.1 mg per assay DMPM, weigh out and dissolve in CHCl₃ 5 mL [³ H]DPPCper assay, place in silanized 13×100 tube add DMPM in CHCl₃ and dry downunder Argon, resuspend in no more than 1 mL water, sonicate at powersetting 10 for 1.5 min., let stand at room temperature for 5-10 min.,adjust the volume to 50 uL per assay, and aliquot into silanized 12×75tubes.

Assay:

Add 50 uL DMPM/DPPC as prepared above, and inhibitor (determine in molefraction, add in water or DMSO), heat to 50° C. for 5-10 min., add waterequilibrated to same temperature if needed, add 150 uL Ca mix (1.2 mL0.12M CaCl₂, 0.6 mL 0.4M HEPES pH 8.0, 10.2 mL water), cool to roomtemperature (15 min.). Start assay by addition of the enzyme, vortexingenzyme into the assay. Quench by addition of 5 uL 5N HCl and extractwith 2×0.5 mL chloroform:methanol (2:1). Dry down extract, resuspend in0.5 mL hexane:ethyl acetate:acetic acid (80:20:1) and apply toconditioned 3 mL Si column (condition with 1 mL hexane). Elute with 2.5mL same solvent into scintillation vials, add 7 mL of the cocktail andcount by any acceptable well known means.

Representative compounds of Formulas (I) and (II) which have shownpositive activity in the above noted assay are:

2-[2-[3-(4-Chloro-3-trifluoromethylphenyl)ureido]-4-trifluoromethylphenoxy]-4,5-dichlorobenzenesulfonic acid;

2-[2-[[[[3,5-Bis(trifluoromethyl)phenyl]amino]carbonyl]amino]-4-chlorophenoxy]-5-chlorobenzenesulfonicacid, and preferably the monosodium salt thereof;

5-Chloro-2-[2-(3-(3,5-di-trifluoromethylphenyl)ureido)-4-trifluoromethylphenyl)thio]benzenesulfonicacid;

5-(1,1-Dimethylpropyl)-2-[4-(trifluoromethyl)-2-[[[[3-(trifluoromethyl)phenyl]amino]carbonyl]amino]-phenoxy]benzenesulfonicacid, and preferably the monosodium salt thereof;

2-[2-[3-(4-Chloro-3-trifluoromethylphenyl)ureido]-4-trifluoromethylphenoxy]-5-(1,1-dimethylpropyl)benzenesulfonicacid;

2-[2-[[[[3,5-Bis(trifluoromethyl)phenyl]amino]carbonyl]amino]-4-(trifluoromethyl)phenoxy]-5-(1,1-dimethylpropyl)benzenesulfonicacid;

5-Chloro-2-[(4-chloro-2-(3-(4-chloro-3-trifluoromethylphenyl)ureido)phenyl)thio]benzenesulfonicacid; and

5-Chloro-2-(4-chloro-2-(2-(3-chlorophenoxy)-5-trifluoromethylphenylaminocarbonylamino)phenyoxybenzenesulfonic acid.

As used herein, various abbreviations and explanations are as follows:[³ H], a molecule that contains tritium atoms, a radioactive isotope;A23187, a compound that allows free entry of calcium into a cell; AA,arachidonic acid; arachidonate, arachidonic acid contained within aphospholipid; free arachidonic acid, arachidonic acid that is notcontained within a phospholipid; [² H₈ ]arachidonic acid, the form ofarachidonic acid labeled with 8 deuterium atoms, a stable isotope;1-alkyl, 1-O-alkyl; 1-alkenyl, 1-O-alk-1'-enyl; BSA, bovine serumalbumin; CoA, coenzyme A; CoA-IT, CoA-independent transacylase; DTr,dithiothreitol; EGTA, [ethylenebis(oxyethylenenitrilo)]tetra aceticacid, a calcium chelator; GPC, sn-glycero-3-phosphocholine; EDTA, ametal ion chelator; GPE, sn-glycero-3-phosphoethanolamine; GC/MS, gaschromatography and mass spectrometry; 5HETE,5(S)-hydroxyeicosa-6,8,11,14-tetraenoic acid; 15HETE,15(S)-hydroxyeicosa-5,8,11,13-tetraenoic acid; HL-60, American TypeTissue Culture designated cell line similar to a monocyte; LTB₄,leukotriene B₄ ; LTC₄, leukotriene C₄ ; LTD₄, leukotriene D₄ ; lyso PAF,1-alkyl-2-lyso-GPC, lyso platelet-activating factor; PLA₂, phospholipaseA₂ ; PBS, phosphate buffered saline; PAF, platelet activating factor,1-alkyl-2-acetyl-GPC; PL, phospholipid; PC, phosphatidylcholine; PE,phosphatilylethanolamine, PI, phosphatidylinositol; PMN,polymorphonuclear neutrophilic cell, neutrophil; PS phosphatidylserine;Rf, the distance a compound travels as a fraction of the solvent front;TLC, thin layer chromatography; U937, American Type Tissue Culturedesignated cell line similar to a monocyte.

The above description fully discloses the invention including preferredembodiments thereof. Modifications and improvements of the embodimentsspecifically disclosed herein are within the scope of the followingclaims. Without further elaboration, it is believed that one skilled inthe art can, using the preceding description, utilize the presentinvention to its fullest extent. Therefore, the Examples herein are tobe construed as merely illustrative and not a limitation of the scope ofthe present invention in any way. The embodiments of the invention inwhich an exclusive property or privilege is claimed are defined asfollows.

What is claimed is:
 1. A method for treating an inflammatory disease ordisorder in a mammal in need thereof which method comprisesadministering to said mammal an effective amount of a compound ofFormula (D ##STR3## wherein R₄ is a phenyl substiuted one to two timesindependently with chlorine or CF₃ ;R₁ is chlorine; R₂ is hydrogen orchlorine; R₃ is chlorine or CF₃ ; provided that when R₁ and R₂ are bothchlorine then R₃ is CF₃ ;and pharmaceutically acceptable salts thereof.2. The method according to claim 1 wherein the inflammatory disease ordisorder is allergic rhinitis, ischemia, reperfusion injury, arthritis,inflammatory bowel disease, Crohn's disease, ulcerative colitis, asthma,adult respiratory distress syndrome, analphylaxis, actinic keratosis,psoriasis, contact dermatitis, or pyresis.
 3. The method according toclaim 1 wherein the inflammatory disease or disorder is mediated bylipid inflammatory mediators, arachidonic acid, its metabolites and/orplatelet activating factor (PAF).
 4. The method according to claim 3wherein the lipid inflammatory meditors are inhibited by the aninhibitor of the enzyme phospholipase A₂ (PLA₂) or Coenzyme Aindependent transacylase (CoA-IT).
 5. The method according to claim 1wherein the compound, or pharmaceutically acceptable salt thereof, is 4.5-Dichloro-2-[4-trifluoromethyl-2-(3-trifluoromethyl-4-chlorophenyl)ureido)phenyl)thio]benzenesulfonicacid;4,5-Dichloro-2-[3-(3,4-dichlorophenyl)ureido]-4-trifluoromethylphenyl)thiobenzenesulfonicacid;5-Chloro-2-[2-(3-(3,5-di-trifluoromethylphenyl)ureido)-4-trifluromethylphenyl)thio]benzenesulfonicacid;5Chloro-2-[(4-chloro-2-((3-(5-chloro-3-trifluoromethylphenyl)ureido)phenyl)thio]benzenesulfonicacid;5-Chloro-2-[(4-chloro-2-[3-(2,4-dichlorophenyl)ureido)phenyl)thio]benzenesulfonicacid; or5-Chloro-2-[(4-chloro-2-(3-(4-chloro-3-trifluoromethylphenyl)ureido)phenyl)thio]benzenesulfonicacid.